A series of respiration-deficient Chinese hamster cell mutants isolated and characterized by our laboratory will be used as starting materials in attempts to isolate and clone nuclear genes coding for proteins in the inner mitochondrial membrane. The initial emphasis is on the genes for succinate dehydrogenase (SDH) and genes affecting complex I of the electron transport chain (NADH-CoQ reductase). Much is to be learned about basic mechanisms in the biogenesis of mammalian mitochondria. We are interested in the coordinate expression and regulation of several nuclear genes, and the detailed mapping of these genes of mammalian chromosomes. Precursor polypeptides made in the cytoplasm have to be recognized, transported across the outer and possibly inner mitochondrial membrane, and then assembled in the inner membrane. Signals on the cytoplasmic precursor polypeptides have to be elucidated, and pathways of post-translational processing have to be worked out. Our approach makes use of the mutants in selections for a complementing, wild-type gene from human cells. The human genes are introduced by somatic cell fusion after irradiation of the human parental cells to introduce extensive chromosome breakage. Thus, in the final hybrid cells, only fragments of the human chromosome I (for SDH), or the X chromosome (for complex I) will be retained. Libraries from such hybrids with less than .1% of the human genome will be constructed and screened for vectors (phage Lambda) with human DNA inserts. In the further analysis a variety of approaches will be tried. Comparisons of independently-derived hybrids will narrow the choice. We have, or are in the process of making, antisera against SDH subunits, which will be used in the identification of genes subcloned into expression vectors and placed into bacteria. Knowledge of a portion of the amino acid sequence of the 70K subunit of SDH will also be exploited; a) to make a specific antiserum, and b) to make synthetic oligonucleotide probes for screening our cloned sequences. With the help of specific antisera against SDH peptides, we also plan to examine further a mutant in our collection which is almost totally defective in mitochondrial protein synthesis and has morphologically abnormal cristae.